Upregulation of nuclear factor E2-related factor 2 (Nrf2) represses the replication of herpes simplex virus type 1

Abstract

Background: Nuclear factor E2-related factor 2 (Nrf2) is an important transcription factor which plays a pivotal role in detoxifying reactive oxygen species (ROS) and has been more recently shown to regulate inflammatory and antiviral responses. However, the role of Nrf2 in Herpes Simplex Virus type 1 (HSV-1) infection is still unclear. In this study, the interaction between the Nrf2 and HSV-1 replication was investigated.

Methods: The levels of oxidative stress was monitored by using 8-hydroxy-2'-deoxyguanosine (8-OHdG) ELISA kits, and the dynamic changes of Nrf2-antioxidant response element (Nrf2-ARE) pathway were detected by Western Blot. The effect of Nrf2-ARE pathway on the regulation of HSV-1 proliferation was analyzed by Western Blot, Real-Time PCR and TCID₅₀ assay.

Results: HSV-1 infection induced oxidative stress. Nrf2 was activated, accompanied by the increase of its down-stream antioxidant enzyme heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1) in the early stage of HSV-1 infection. The proliferation of HSV-1 was inhibited by overexpression of Nrf2 or treatment with its activator tert-Butylhydroquinone (tBHQ). On the contrary, silencing of Nrf2 promotes virus replication. HO-1 is involved in the regulation of IFN response, leading to efficient anti-HSV-1 effects.

Conclusion: Our observations indicate that the Nrf2-ARE pathway activates a passive defensive response in the early stage of HSV-1 infection. Targeting the Nrf2 pathway demonstrates the potential for combating HSV-1 infection.

Keywords: Heme oxygenase-1 (HO-1); Herpes simplex virus type 1 (HSV-1); NAD(P)H quinone oxidoreductase 1 (NQO1); Nuclear factor E2-related factor 2 (Nrf2); Oxidative stress.

Fig. 1

HSV-1 infection induced oxidative stress and Nrf2 was activated in the early stage of HSV-1 infection. a HEp-2 cells were infected with HSV-1 at an MOI of 1 and samples were collected to identify the oxidative stress by 8-OHdG ELISA kit at 0, 6, 12 and 24 hpi. b The expression of Nrf2, Keap1, HO-1, NQO1 and virus protein ICP4 were identified by Western Blot. c The expression of Nrf2-ARE pathway protein was detected after infected with HSV-1 at different MOIs by Western Blot. Data represent the results from three independent experiments (Mean ± SD, ***P < 0.001, ns, not significant)

Fig. 2

Ectopic expression of Nrf2 dampened HSV-1 replication. HEp-2 cells were pretreated with two different concentrations (1, 0.5 µg) of Nrf2 plasmid 24 h before HSV-1 (MOI = 1) infection, HEp-2 cells infected HSV-1 were used as positive control and untreated HEp-2 cells were used as negative control. Cells were cultured in maintenance medium for 24 h, then protein and total RNA were extracted. a The expression of Nrf2, HO-1, NQO1 and virus protein gD were identified by Western Blot. b Cells were repeatedly frozen and thawed for 3 times, then the viral titer of the supernatant was measured by TCID₅₀ assay. c The mRNA level of IFN-α, OAS1 and ISG15 were detected by Real-Time PCR. Data represent the results from three independent experiments (Mean ± SD, **P < 0.01, ***P < 0.001, ns, not significant)

Fig. 3

Nrf2 agonist tBHQ inhibit HSV-1 replication. HEp-2 cells were pretreated with two different concentrations (10 µM, 25 µM) of tBHQ 12 h before HSV-1 (MOI = 1) infection, HEp-2 cells infected HSV-1 were used as positive control and untreated HEp-2 cells were used as negative control. Cells were cultured in maintenance medium for 24 h, then protein and total RNA were extracted. a The expression of Nrf2, HO-1, NQO1 and virus protein gD were identified by Western Blot. b Cells were repeatedly frozen and thawed for 3 times, then the viral titer of the supernatant was measured by TCID₅₀ assay. c The mRNA level of IFN-α, OAS1 and ISG15 were detected by Real-Time PCR. Data represent the results from three independent experiments (Mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant)

Fig. 4

Silencing of Nrf2 promotes virus replication. HEp-2 cells were pretreated with Nrf2 siRNA (100 nM) or NC siRNA (100 nM) 12 h before HSV-1 (MOI = 1) infection, HEp-2 cells infected HSV-1 were used as positive control and untreated HEp-2 cells were used as negative control. Cells were cultured in maintenance medium for 24 h, then protein and total RNA were extracted. a The expression of Nrf2, HO-1, NQO1 and virus protein gD were identified by Western Blot. b Cells were repeatedly frozen and thawed for 3 times, then the viral titer of the supernatant was measured by TCID₅₀ assay. Data represent the results from three independent experiments (Mean ± SD, **P < 0.01, ns, not significant)